Methods of blocking tissue destruction by autoreactive t cells

ABSTRACT

Methods for blocking autoreactive T cell-initiated destruction of tissues in a mammal are provided. In one embodiment, the method comprises administering a purified CD24 polypeptide, a fusion protein comprising such polypeptide, or a biologically active fragment of such polypeptide to a mammalian subject who is suspected of having or predisposed to having an autoimmune disease. In another embodiment, anti-CD24 antibody or anti-CD24 Fab fragments are administered to the subject. In another embodiment, the method comprises administering a CD24 antisense molecule, an expression vector encoding a CD24 antisense molecule, CD24 dsRNAi, or an expression vector encoding CD24 dsRNAi to the subject. The present invention also relates to isolated and purified CD24 fusion proteins employed in the present methods and to transgenic mice that express the human CD24 protein on their T cells and/or their vascular endothelial cells but do not express murine heat shock antigen on any cells.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in part of U.S. application Ser. No.09/822,851 which was filed on Mar. 29, 2001, and claims priority fromU.S. Provisional Patent Application No. 60/192,814, filed on Mar. 29,2000.

STATEMENT OF GOVERNMENT SUPPORT

This invention is supported, at least in part, by Grant No. AI32981 fromthe National Institute of Health, USA. The U.S. government has certainrights in this invention.

BACKGROUND OF THE INVENTION

The present invention relates to agents and methods for blockingdeleterious T cell mediated immune reactions. Such reactions occur inautoimmune diseases, such as for example, multiple sclerosis (MS),rheumatoid arthritis, systemic lupus erythematosis, psoriasis, diabetes,and allergies. Such reactions also occur during rejection oftransplants.

In theory, autoimmune diseases can be prevented by blocking activationof T cells and formation of autoreactive T cells. Accordingly, there area number of studies being conducted to identify methods or agents thatcan be used to block activation of T cells (J Clin Invest. 1995 June;95(6):2783-9; J Med Chem. 2002 Jan. 17; 45(2):275-83). Unfortunately,since patients with autoimmune diseases have already developedautoreactive T cells, these methods have limited value for treatment ofautoimmune diseases. Moreover, agents that prevent systemic T cellactivation often cause serious side effects. For example, treatment withagents that block activation of T cells can also render the patient moresusceptible to infections and cancer. Thus, it is desirable to have newmethods for treating autoimmune diseases. A method which reduces thedestruction of targeted tissues that is initiated by autoreactive Tcells is especially desirable.

SUMMARY OF THE INVENTION

The present invention provides methods for blocking or reducingautoreactive T cell-initiated destruction of tissues in a mammal Themethods employ an agent that inhibits or reduces interaction of the CD24polypeptide with its functional ligand. The CD24 polypeptide is found onthe cell membrane of activated T cells and other cell types, such as Bcells, dendritic cells, epithelial cells and vascular endothelial cells.

In one embodiment, the method comprises administering a pharmaceuticalcomposition comprising a biologically effective amount of an isolatedand purified polypeptide, referred to hereinafter as the “HSA/CD24”polypeptide, a fusion protein comprising the HSA/CD24 polypeptidepolypeptide, or a biologically active fragment of the HSA/CD24polypeptide to a mammal in need of the same, i.e., a mammal who issuspected of having, known to have, or predisposed to have an autoimmunedisease. As used herein, “mammal” refers to rats, mice, cats, dogs,cows, pigs, rabbits, and primates. Exemplary primates include monkeys,chimpanzees, and humans. As used herein the term “HSA/CD24” refers notonly to the protein portion of the heat stable antigen (HSA) found onthe surface of mouse cells but also to the mammalian homologs of mouseHSA. Thus, the term “HSA/CD24”, as used in the present application,encompasses the polypeptide portion of human CD24 and rat CD24, theknown human and rat homologs of mouse HSA. Preferably, the HSA/CD24polypeptide is glycosylated. The fusion protein comprises the HSA/CD24polypeptide or a truncated form of the HSA/CD24 linked by a peptide bondto a peptide or protein tag. In a preferred embodiment, the HSA/CD24fragment comprises the core region of the HSA/CD24 polypeptide.

In another embodiment, the method comprises administering apharmaceutical composition comprising a biologically effective amount ofan anti- HSA/CD24 antibody or anti-HSA/CD24 Fab fragments to a mammalknown to have, suspected of having, or predisposed to having anautoimmune disease.

In another aspect, the method comprises administering to the subject anagent that reduces expression of the CD24 polypeptide in T cells. Suchmethods employ agents that disrupt the function of the CD24 gene at thegenomic, transcriptional, post-transcriptional and translational levels.In one embodiment, the agent is an antisense molecule (referred tohereinafter as “CD24 antisense”) which reduces transcription of the CD24gene or translation of the CD24 gene transcript in autoreactive T cells.In another embodiment, the agent is a double stranded RNA molecule(referred to hereinafter as “CD24 dsRNAi”) which interferes withexpression of the CD24 gene.

The present invention also relates to a method of blocking binding ofautoreactive T cells to vascular endothelial cells. In one aspect, themethod comprises contacting the vascular endothelial cells with asufficient amount of an HSA/CD24 polypeptide or a fragment thereof, or afusion protein comprising HSA/CD24 polypeptide or a fragment thereof, oranti-HSA antibodies to inhibit interaction of the autoreactive T cellswith the vascular endothelial cells. In another aspect, the methodcomprises introducing an oligonucleotide or polynucleotide that inhibitsexpression of the CD24 polypeptide into the autoreactive T cell, or thevascular endothelial cell or both. Examples of such oligonucleotides andpolynucleotides include, but are not limited to, a CD24 antisenseoligonucleotide, an expression vector comprising a polynucleotide ornucleic acid encoding a CD24 antisense oligonucleotide, a CD24 dsRNAi,and an expression vector comprising a polynucleotide or nucleic acidencoding a CD24 dsRNAi.

The present invention also relates to isolated and purified HSA/CD24fusion proteins employed in the above-described methods and totransgenic or knock in mice that express the human CD24 protein on theirT cells or their vascular endothelial cells or all other cell types thatnormally express CD24 but, as a result of targeted mutation, do notexpress murine HSA on any cells. Such mice provide a unique model totest the effectiveness of drugs designed to block or enhance thebiological function of human CD24-mediated autoimmune diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Targeted mutations of HSA and CD28 reveal two distinctcheckpoints in the development of EAE. a. Targeted mutations of eitherHSA or CD28 prevent induction of EAE. WT, CD28(−/−) or HSA(−/−) micewere immunized with MOG peptide. Clinical signs were scored as describedin the method section. b. Proliferative response of lymph node T cellsto MOG peptides. Draining lymph node cells from day 10-immunized micewere stimulated with given concentrations of MOG peptide and irradiatedsyngeneic naive spleen cells as antigen-presenting cells. c. Enumerationof cytokine-producers by ELISpot. Draining lymph node cells used in bwere used as responder cells. The numbers of cells secreting either IL2,IL4, and IFNγ among 1×10⁶ lymph node cells in response to MOG peptide(AA35-55) were presented. Data shown were means+/−SEM from threeindependent experiments.

FIG. 2. Histological analysis of spinal cord of MOG immunized WT orHSA(−/−) mice. a: The means and SEM of histological scores of WT andHSA(−/−) mice spinal cords. Ten independent cross sections, fromcervical to sacral regions, were examined in each spinal cord. The dataare summarized from 30 spinal cord sections from 3 mice in each group.b. Representative histology in immunized WT mice, all sections examinedcontain histology lesions. c and d. Histology sections (100×) ofimmunized HSA(−/−) mice. A lesion-free section is presented in c, whilea lesion containing section is presented in d.

FIG. 3. Requirement for HSA expression on both T cells and non-T hostcells for the induction of EAR. Histology (63× for a, b, c and the leftpanel of d; 200x for the right panel of d) of spinal cords of theHSA(−/−)(a, b) or WT(c, d) recipient mice on day 12 after adoptivetransfer. Draining lymph node cells were isolated from either WT orHSA(/−) mice after immunization, and were stimulated with antigen andIL2 for 4 days in vitro. The activated T cells were injected into eitherWT or HSA(−/−) mice (100×10⁶ cells per mouse). EAE development wasmonitored daily for clinical signs. At 12 days after transfer, recipientmice were sacrificed and spinal cords were processed for histologicalexamination. No disease was observed in WT>HSA(−/−), HSA(−/−)>WT, orHSA(−/)>HSA(−/−) recipients.

FIG. 4. Clinical scores of the adoptive transfer experiment with 4(WT>HSA(−/−) and HSA(−/−)>WT groups) or 5 (WT>WT and HSA(−/−)>HSA(−/−)groups) mice per group.

FIG. 5. Transgenic expression of HSA exclusively on T cell lineage isinsufficient for EAE development. a. Phenotypes of WT, HSA-TG, HSA(−/−),and HSATG/HSA(−/−) mice by flow cytometry using anti-HSA and anti-CD3mAbs. b. EAE score in WT, HSATG, HSA(−/−), and HSATG/HSA(−/−) mice afterimmunization with the MOG peptides.

FIG. 6. HSAIg ameliorates EAE a. Analysis of HSAIg by SDS-PAGE. 10 μg ofpurified HSAIg was separated by 10% reducing (R) and non-reducingSDS-PAGE. The proteins were stained by Comassie blue. The EAR score forcontrol (PBS) or HSAIg-treated mice. EAE was induced in WT mice asdescribed in Materials and Methods. On days 8, 10, 12, 14 and 22 afterimmunization, five mice per group were injected (i.p.) with 100 μg/mouseof either HSAlg or 100 ml of PBS as control. The effect of HSAIg hasbeen evaluated in three independent experiments with similar results.

FIG. 7 shows the amino acid sequence, SEQ ID NO. 1, of the mouse HSApolypeptide. The signal peptide extends from amino acid 1 through aminoacid 26 of the sequence. The glycophosphatidyl (GPI) anchor regionincludes and extends from amino acid 54 through amino acid 76.

FIG. 8 shows the amino acid sequence, SEQ ID NO. 2, of the human CD24polypeptide. The signal peptide extends from amino acid 1 through aminoacid 26 of the sequence. The glycophosphatidyl (GPI) anchor regionincludes and extends from amino acid 60 through amino acid 80.

FIG. 9 shows the amino acid sequence, SEQ ID NO. 3, of the rat CD24polypeptide. The signal peptide extends from amino acid 1 through aminoacid 26 of the sequence. The glycophosphatidyl (GPI) anchor regionincludes and extends from amino acid 57 through amino acid 76.

FIG. 10 shows the DNA sequence, SEQ ID NO. 4, of a fusion gene whichcomprises a nucleotide sequence encoding HSA fused to the genomicsequence of human IgG1 Fc. The predicted sequence of the cDNA, SEQ IDNO. 5, which results from splicing of the introns IgG1 Fc sequence andthe predicted amino acid sequence, SEQ ID NO. 14, are also shown in thisfigure. The normal font with under line is HSA sequence, bold phase isnew sequence, italics is IgG1 Fc sequence.

FIG. 11 is a diagram of a construct for producing human CD24 geneknock-in mice. Arm 1 of the knock in construct comprises nucleotide 2001through nucleotide 5500 of the mouse HSA/CD14 gene, GenBank AccessionNo. X72910. Arm 2 of the construct is a chimera gene consisting of thelast 256 by sequence of exon 1 of the mouse CD24 gene, the first 240 bysequence of exon 2 of the human CD24 gene and about 3 kb of mouse CD24sequence which comprises remaining exon 2 sequence encoding for 3′untranslated region and 3′ sequence of the mouse CD24 gene (Seq I.D.22).

FIG. 12 is a diagram of a plasmid for producing transgenic miceexpressing human CD24 in T cells. To produce this plasmid human CD24coding sequence is subcloned into transgenic construct vector p1017 BamHI site, which is described in EMBO J. 9: 3821-3829, 1990) CD24 forwardprimer (CD24F.Bam): G GCC GGA TCC ATG GGC AGA GCA ATG GTG with BamHlsite 5′ to ATG start codon. CD24 reverse primer (CD24R. XhoBam): G GCCGGA TCC CTC GAG TTA AGA GTA GAG ATG CAG with Barn HI and Xho I sites 3′to TAA stop codon.

FIG. 13 is a diagram of a vector for producing mice that express CD24 invascular endothelial cells.

FIG. 14 shows a comparison of mouse and human CD24 cDNA sequences, andthe preferred sequences to be targeted by CD24 antisense RNA and CD24dsRNAi . Human CD24 and mouse CD24 cDNA sequences (Human CD24(XM_(—)099027) and Mouse CD24 (NM_(—)009846)) are aligned by doubleblast search. The regions with a stretch of identity that are 17 or morebase pairs in length are highlighted as preferred targets for CD24antisense and dsRNAi agents.

FIG. 15 is a dot plot demonstrating inhibition of CD24 expression in CHOcells using dsRNAi. Chinese hamster ovary (CHO) cells were transientlytransfected with human CD24 plasmid alone (top), the CD24 plasmid plusdsRNAi (middle) or CD24 plasmid plus inverted dsRNA as control (lowerpanel). The expression of CD24 on cell surface was analyzed at 72 hoursafter transfection using phycoerythorin-conjugated anti-human CD24 mAb(BD Pharmingen). Note essential absence of CD24-expression in cellstreated with CD24 dsRNAi.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for blocking destruction oftissue by autoreactive T cells in a mammalian subject. The methodsemploy agents which inhibit or reduce, either directly or indirectly,the interaction of the CD24 polypeptide with its functional ligand. TheCD 24 polypeptide is present on the cell membrane of activated T cellsand other cell types such as B cells, dendritic cells, epithelial cellsand vascular endothelial cells.

In one embodiment, the method comprises administering a pharmaceuticalcomposition comprising a biologically effective amount of an isolatedand purified HSA/CD24 polypeptide or a fragment thereof to a mammalsuspected of having an autoimmune disease. In another embodiment afusion protein comprising the HSA/CD24 polypeptide or fragment thereoflinked by a peptide bond to a peptide or protein tag is administered tothe mammal. Preferably, the HSA/CD24 polypeptide is glycosylated. Inanother embodiment an antibody which is immunospecific for the HSA/CD24polypeptide is administered to the mammal.

In another aspect, the method comprises administering to the subject anagent that reduces expression of the CD24 polypeptide in T cells. Suchmethods employ agents that disrupt the function of the CD24 gene at thegenomic, transcriptional, post-transcriptional and translational levels.In this aspect, embodiments of the present method employ antisensemolecules or dsRNAi to inhibit expression of the CD24/HSA gene andproduction of the CD24 polypeptide in the autoreactive T cells of themammalian subject

The present invention also relates to a method of treating a humansubject known to have, suspected of having, or predisposed to having anautoimmune disease. The methods involve treating the human subject withan agent which inhibit or reduce, either directly or indirectly, theinteraction of the CD24 polypeptide which is present on the cell surfaceof the subject's cells, including but not limited to activated T cells,with the functional ligand of CD24. In accordance with the presentinvention, it is believed that such ligands include, but are not limitedto, CD24 itself, P-selectin, and very late antigen 4.

In one aspect, the therapeutic method comprises administering apharmaceutical composition comprising a biologically effective amount ofan isolated and purified human CD24 polypeptide or fragment thereof, ora fusion protein comprising such molecule, to the human subject. Inanother embodiment of this therapeutic method, the pharmaceuticalcomposition comprises anti-human CD24 antibodies or their Fab fragments.Preferably, the anti-human CD24 antibody is a monoclonal antibody, morepreferably a humanized monoclonal anti-human CD24 antibody. Preferably,the pharmaceutical composition is administered after autoreactive Tcells have been detected in the human subject.

Preferably, the pharmaceutical composition is administered by injection.The present method is useful for treating subjects suspected of havingautoimmune diseases such as for example, multiple sclerosis (MS),rheumatoid arthritis, and insulin-dependent diabetes mellitus. By“treating” is meant ameliorating or tempering the severity of thecondition, either occurring or expected to occur in the future. In casesof autoimmune demyelinating diseases of the CNS such as for example MS,the pharmaceutical composition is administered either when patients haveclinical symptoms, or when they are in temporary remission. Preferably,the protocol involves intravenous injection. In the case of rheumatoidarthritis, the pharmaceutical composition, preferably, is administeredintravenously (i.v.) after the acute symptoms are relieved by othertherapeutic methods. In the case of insulin dependent diabetes mellitus,the pharmaceutical composition, preferably, is administeredintravenously after autoreactive T cells are detected in the peripheralblood.

Further embodiments of this therapeutic method employ antisensemolecules or dsRNAi to inhibit expression of the CD24 gene andproduction of CD24 polypeptide in the T cells of the human subject.

Pharmaceutical Composition

The pharmaceutical composition comprises a biologically effective amountof an HSA/CD24 polypeptide or a biologically active variant thereof oralternatively a fragment of an HSA/CD24 polypeptide or a biologicallyactive variant thereof, and preferably a relatively inert topicalcarrier. Many such carriers are routinely used and can be identified byreference to pharmaceutical texts.

HSA Antigen

The mouse HSA antigen and the mammalian homologs thereof arepolypeptides comprising approximately 76-80 amino acids. The HSApolypeptide and the mammalian homologs thereof are cell surfacemolecules which are linked to the cell membrane via aglycophosphatidylinositol (GPI) tail. The HSA antigen is constitutivelyexpressed on most hematopoietic and developing neuronal cells. In somelymphocytes, such as for example T cells, expression of the HSApolypeptide is induced. As shown in FIGS. 7-9, the immature forms ofmouse HSA antigen, human CD24 and rat CD24 comprise a signal sequence, acore region that is maintained in the mature protein, and a GPI anchorregion. As shown in FIG. 7, the signal sequence of the mouse HSA antigenincludes amino acid 1 through amino acid 26 of SEQ ID NO. 1; the coreregion includes amino acid 27 through amino acid 53 of SEQ ID NO. 1; andthe GPI region includes amino acid 54 through amino acid 75 of SEQ IDNO. 1. As shown in FIG. 8, the signal sequence of the human CD24 antigenincludes amino acid 1 through amino acid 26 of SEQ ID NO.2; the coreregion includes amino acid 27 through amino acid 59 of SEQ ID NO. 2 andthe GPI region includes amino acid 60 through amino acid 80 of SEQ IDNO. 2. As shown in FIG. 9, the signal sequence of the rat CD24 antigenincludes amino acid 1 through amino acid 26 of SEQ ID NO.3; the coreregion includes amino acid 27 through amino acid 56 of SEQ ID NO. 3 andthe GPI region includes amino acid 57 through amino acid 76 of SEQ IDNO. 3. The nucleotide sequence of a polynucleotide which encodes thehuman CD4 polypeptide is available at the GenBank Accession No.AK000168. The nucleotide sequence of a cDNA which encodes the rat CD24polypeptide is available at GenBank Accession No. AWK12164. Thenucleotide sequence of a cDNA which encodes mouse HSA antigen isavailable at GenBank Accession M58661.

The present invention relates to novel method of using an HSA/CD24 polypeptide or fragment thereof to treat autoimmune diseases. Preferably, thepolypeptide or fragment is glycosylated. In one embodiment the HSA/CD24fragment is a truncated form of the HSA/CD24 polypeptide which lacks afew amino acids, i.e., from 1 to 2 amino acids, at the amino terminus orcarboxy terminus thereof. In another embodiment the HSA/CD24 fragment isa polypeptide which comprises essentially only the core region of theHSA/CD24 polypeptide, i.e. the HSA/CD24 fragment lacks most or all ofthe signal peptide and most or all of the CPI anchor region. As usedherein the term HSA/CD24 polypeptide comprises all mammalian homologs ofmouse HSA , including human CD24 and rat CD24.

The HSA/CD24 polypeptide or HSA/CD24 fragment that is used in thepharmaceutical composition is the naturally-occurring HSA/CD24polypeptide, a biologically active fragment of the naturally-occurringHSA/CD24 polypeptide, a biologically active variant of thenaturally-occurring HSA/CD24 polypeptide, or a biologically activevariant of a fragment of the naturally-occurring HSA/CD24 polypeptideThe biologically active variant of the HSA/CD24 polypeptide has an aminoacid sequence which is at least 80%, more preferably at least 93%, mostpreferably at least 96% identical to the amino acid sequence of thenaturally occurring HSA/CD24 polypeptide that is present in the mammalto whom the pharmaceutical composition is being administered. Similarly,the biologically active variant of the fragment of the HSA/CD24polypeptide has an amino acid sequence which is at least 80%, preferablyat least 90%, more preferably at least 95% identical to the amino acidsequence of the corresponding naturally-occurring HSA/CD24 fragment. Formurine HSA, alteration in Positions 1(Asn), 4(Ser), 13(Asn), 15(Ser),17(Ser), 21(Ser), 22(Asn), 24(Thr) and 25(Thr) of the mature peptide,i.e., the peptide which lacks the signal peptide, may alterglycosylation and interfere with its ability to block destruction oftissue by autoreactive T cells. Thus, it is preferred that alternationsnot be made at these sites. In the human homologue of HSA, i.e., humanCD24, 20 out of 31 amino acids are potential glycosylation sites.

An HSA/CD24 polypeptide which is less than 100% identical to thenaturally occurring HSA/CD24 polypeptide has an altered sequence inwhich one or more of the amino acids in the HSA homologue is deleted orsubstituted, or one or more amino acids are inserted into the sequenceof the naturally occurring HSA/CD24 polypeptide. HSA/CD24 sequenceswhich are at least 95% identical to the naturally occurring HSA/CD24sequence have no more than 5 alterations, i.e., any combination ofdeletions, insertions or substitutions, per 100 amino acids of thereference sequence. Percent identity is determined by comparing theamino acid sequence of the altered HSA/CD24 sequence with the naturallyoccurring sequence using MEGALIGN project in the DNA STAR program.Sequences are aligned for identity calculations using the method of thesoftware basic local alignment search tool in the BLAST network service(the National Center for Biotechnology Information, Bethesda, MD) whichemploys the method of Altschul, S. F., Gish, W., Miller, W., Myers, E.W. & Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410. Identities arecalculated by the Align program (DNAstar, Inc.) In all cases, internalgaps and amino acid insertions in the candidate sequence as aligned arenot ignored when making the identity calculation.

While it is possible to have nonconservative amino acid substitutions,it is preferred that the substitutions be conservative amino acidsubstitutions, in which the substituted amino acid has similarstructural or chemical properties with the corresponding amino acid inthe reference sequence. By way of example, conservative amino acidsubstitutions involve substitution of one aliphatic or hydrophobic aminoacids, e.g. alanine, valine, leucine and isoleucine, with another;substitution of one hydroxyl-containing amino acid, e.g. serine andthreonine, with another; substitution of one acidic residue, e.g.glutamic acid or aspartic acid, with another; replacement of oneamide-containing residue, e.g. asparagine and glutamine, with another;replacement of one aromatic, residue, e.g. phenylalanine and tyrosine,with another; replacement of one basic residue, e.g. lysine, arginineand histidine, with another; and replacement of one small amino acid,e.g., alanine, serine, threonine, methionine, and glycine, with another.

The biologically active fragments and variants of a naturally-occurringHSA/CD24 have an ID₅₀ which is comparable to, i.e., not more than twicethe value of, the ID₅₀ of the corresponding naturally- occurringHSA/CD24 polypeptide. The ID₅₀ of the HSA/CD24 variant or fragment andits corresponding naturally-occurring polypeptide is determined bymeasuring the amount of these polypeptides needed to reduce the clinicalsymptoms in experimental autoimmune models, such as EAE or Type IIdiabetes in NOD mouse or rat. Alternatively, one can determine the ID₅₀of the biologically active HSA/CD24 variant or fragment and itscorresponding naturally occurring HSA/CD24 polypeptide by an adhesionassay or assays that measure migration of T cells through endothelialcell monolayer in transwell culture. The amount of the biologicallyactive variant of the HSA/CD24 polypeptide or fragment thereof needed toreduce binding of activated T cells to vascular endothelial cells by atleast 50%, preferably, is no greater than, twice the amount of thecorresponding naturally occurring HSA/CD24 polypeptide or fragmentthereof.

The present method also employs fusion proteins comprising an HSA/CD24polypeptide or a biologically active fragment thereof and a tag, i.e.,or one or more amino acids, preferably from about 5 to 300 amino acidswhich are added to the amino terminus of, the carboxy terminus of, orany point within the amino acid sequence of the HSA/CD24 polypeptide orthe biologically active fragment thereof. Preferably, the HSA/CD24polypeptide or core region thereof is glycosylated. Typically, suchadditions are made to simplify purification of an expressed recombinantform of the corresponding HSA/CD24 polypeptide or core region thereof.Such tags are known in the art. Representative examples of such tagsinclude sequences which encode a series of histidine residues, theepitope tag FLAG, the Herpes simplex glycoprotein D, beta-galactosidase,maltose binding protein, or glutathione S-transferase. Preferably, thefusion protein comprises the HSA polypeptide or a fragment thereoflinked by a peptide bond to the hinge-CH2-CH3 regions of humanimmunoglobin G1 (“IgG1”). The fusion protein can be easily purified byaffinity chromatography using either anti-IgG or protein A or protein G.Since IgG is not immunogenic in humans, the fusion protein can beadministrated repeatedly if necessary.

Methods of Preparing the HSA/CD24 Polypeptide or Fusion Protein

The HSA/CD24 polypeptides and fusion proteins may be produced by usingcell-free translation systems and RNA molecules derived from DNAconstructs that encode the polypeptide or fusion protein. Preferably,the HSA/CD24 polypeptide or fusion protein is made by transfecting hostcells with expression vectors that comprise a DNA sequence that encodesthe respective HSA/CD24 polypeptide or fusion protein and then inducingexpression of the polypeptide in the host cells. For recombinantproduction, recombinant constructs comprising one or more of thesequences which encode the HSA/CD24 polypeptide or fusion protein areintroduced into host cells by conventional methods such as calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape lading, ballistic introduction orinfection.

The HSA/CD24 polypeptide or fusion protein may be expressed in suitablehost cells, such as for example, mammalian cells, yeast, insect cells orother cells under the control of appropriate promoters usingconventional techniques. Suitable hosts include, but are not limited to,CHO, COS cells and 293 HEK cells. Following transformation of thesuitable host strain and growth of the host strain to an appropriatecell density, the cells are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification of the epitope or chimeric peptide. For obtainingproperly glycosylated forms of the protein, it is preferred that CHOcells be used.

Conventional procedures for isolating recombinant proteins fromtransformed host cells, such as isolation by initial extraction fromcell pellets or from cell culture medium, followed by salting-out, andone or more chromatography steps, including aqueous ion exchangechromatography, size exclusion chromatography steps, and highperformance liquid chromatography (HPLC), and affinity chromatographymay be used to isolate the recombinant polypeptide.

Carrier

The acceptable carrier is a physiologically acceptable diluent oradjuvant. The term physiologically acceptable means a non-toxic materialthat does not interfere with the effectiveness of HSA. Thecharacteristics of the carrier will depend on the route ofadministration and particular compound or combination of compounds inthe composition. Preparation of such formulations is within the level ofskill in the art. The composition may further contain other agents whicheither enhance the activity of the HSA or complement its activity. Thecomposition may further comprise fillers, salts, buffers, stabilizers,solubilizers, and other materials well known in the art.

Dosage

A biologically effective amount is an amount sufficient to partially orcompletely block destruction of the targeted tissue initiated by theautoreactive T cell or to ameliorate the pathological effects of theautoimmune disease. The effective amount can be achieved by oneadministration of the composition. Alternatively, the effective amountis achieved by multiple administration of the composition to the mammal.

Antibodies

The invention further provides a therapeutic method which comprisesadministering a pharmaceutically effective amount of an anti-HSA/CD24antibody, preferably a humanized anti-HSA/CD24 antibody, to a humansubject suspected of having an autoimmune disease. The anti-HSA/CD24antibody is immunospecific for the HSA/CD24 polypeptide meaning theantibody has substantially greater affinity for the HSA/CD24 polypeptidethan for other polypeptides that are found on the T cells of the mammalbeing treated. Various forms of an anti- HSA/CD24 antibody may be usedin this therapeutic method. For example, the anti-HSA/CD24 antibody maybe a full length antibody (e.g., having a human immunoglobulin constantregion) or an antibody fragment (e.g. a F(ab′)₂).

The term “antibody” as used herein encompasses monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments so long as they exhibit the desired biological activity.“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., Nature 256: 495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature 352: 624-628 (1991) andMarks et al., J. Mol. Biol. 222: 581-597 (1991), for example.

The monoclonal antibodies herein include “chimeric” antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step

In order to avoid potential immunogenicity of the mAbs in human, themAbs that have desired function are preferably humanized. “Humanized”forms of non-human (e.g., murine) antibodies are chimeric antibodieswhich contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which hypervariable region residues of therecipient are replaced by hypervariable region residues from a non-humanspecies (donor antibody) such as mouse, rat, rabbit or nonhuman primatehaving the desired specificity, affinity, and capacity. In someinstances, Fv framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues which are not found in the recipientantibody or in the donor antibody. These modifications are made tofurther refine antibody performance. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

Alternatively, transgenic mice with human IgV and IgC genes may be usedto produce human mAb specific for human CD24. These mice are availablefrom Abgenix, Inc., and Mederex, Inc, and the art has been describedfully (Nature Genetics, 1997, 15: 146).

Antisense Molecules

In certain aspects, the present therapeutic methods employ agents whichreduce or inhibit expression of the CD24 gene or production of the CD24polypeptide in the T cells of human subjects known to have or suspectedof having an autoimmune disease, such as multiple sclerosis, rheumatoidarthritis, and type II diabetes.

One such agent is an antisense molecule. The antisense molecule for CD24is an oligomer which comprises from 20 to 200 bases, preferably lessthan 100 bases, and is targeted to a nucleic acid encoding the humanCD24 polypeptide, in other words, the human CD24 gene or mRNA expressedfrom the human CD24 gene. The targeting process involves determinationof a site or sites within the nucleic acid sequence of the CD24 gene ormRNA for the oligonucleotide interaction to occur such thattranscription of the CD24 gene or translation of the CD24 mRNA will bereduced in the human subject's T cells. Once the target site or siteshave been identified, oligonucleotides are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired reduction in expression ofthe CD24 polypeptide. Such inhibition can be measured in ways which areroutine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression, or flowcytometry analysis for cell surface expression.

“Hybridization”, as used herein means hydrogen bonding, also known asWatson-Crick base pairing, between complementary bases, usually onopposite nucleic acid strands or two regions of a nucleic acid strand.“Specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the DNA or RNA target and theoligonucleotide. It is understood that an oligonucleotide need not be100% complementary to its target nucleic acid sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when binding of the oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of utility, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment or, in the case of in vitro assays, underconditions in which the assays are conducted. Affinity of anoligonucleotide for its target (in this case a nucleic acid encodingCD24 polypeptide) is routinely determined by measuring the Tm of anoligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target.

In the context of this therapeutic method, the term “oligonucleotide”refers to an oligomer or polymer of nucleotide or nucleoside monomersconsisting of naturally occurring bases, sugars and intersugar(backbone) linkages. The term “oligonucleotide” also includes oligomerscomprising non-naturally occurring monomers, or portions thereof, whichfunction similarly. Modifications may be on one or more bases, sugars,or backbone linkages, or combinations of these; such modifications arewell known in the art. Modified or substituted oligonucleotides areoften preferred over native forms because of properties such as, forexample, enhanced cellular uptake and increased stability in thepresence of nucleases. A number of nucleotide and nucleosidemodifications have been shown to make the oligonucleotide into whichthey are incorporated more resistant to nuclease digestion than thenative oligodeoxynucleotide. Nuclease resistance is routinely measuredby incubating oligonucleotides with cellular extracts or isolatednuclease solutions and measuring the extent of intact oligonucleotideremaining over time, usually by gel electrophoresis. Oligonucleotideswhich have been modified to enhance their nuclease resistance surviveintact for a longer time than unmodified oligonucleotides. A variety ofoligonucleotide modifications have been demonstrated to enhance orconfer nuclease resistance. In some cases, oligonucleotide modificationswhich enhance target binding affinity are also, independently, able toenhance nuclease resistance. A discussion of antisense oligonucleotidesand some desirable modifications can be found in De Mesmaeker et al.Acc. Chem. Res. 1995, 28, 366-374.

Specific examples of some oligonucleotides contemplated for the presentmethod include those containing modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. The oligonucleotides may be chimericoligonucleotides. “Chimeric oligonucleotides” as used herein meanoligonucleotides which contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the RNA target) and a region that is a substratefor RNase H cleavage. In one embodiment, a chimeric oligonucleotidecomprises at least one region modified to increase target bindingaffinity, and, usually, a region that acts as a substrate for RNAse H.RNAse H is a cellular endonuclease that cleaves the RNA strand ofRNA:DNA duplexes; activation of this enzyme therefore results incleavage of the RNA target, and thus can greatly enhance the efficiencyof antisense inhibition. Cleavage of the RNA target can be routinelydemonstrated by gel electrophoresis.

Alternatively, an expression vector comprising a polynucleotide ornucleic acid encoding an antisense oligonucleotide targeted to nucleicacids that encode the CD24 polypeptide are introduced into the subject'sT cells. The CD24 antisense encoding nucleic acid is operatively linkedto a promoter. A “promoter” is a sequence that directs the binding ofRNA polymerase and thereby promotes RNA synthesis, which in the presentmethod is synthesis of the antisense oligonucleotide. Operatively linkedis understood to mean that the CD24 antisense encoding sequence isjoined to the promoter region such that the promoter is oriented 5′ tothe CD24 antisense encoding sequence and is of an appropriate distancefrom the transcription start site, so that the transcription of thepolynucleotide which encodes the CD24 antisense oligonucleotide will bedependent on or controlled by the promoter sequence. The arts ofrestriction enzyme digestion and nucleic acid ligation to be used inconstruction of the CD24 antisense encoding polynucleotide-promoterconstruct are well known in the art as exemplified by Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y., 1982,(incorporated herein by reference). Many examples of constitutivepromoters have been described in the art such as those isolated fromcytomegalovirus early gene, murine MHC class I, actin, etc. An exampleof T-cell specific promoter is the lck promoter (EMBO J. 9:3821-3829,1990), which we have shown to be able to drive T cell-specificexpression (Eur. J. Immunol 27:2524-2528, 1997) . An example of vascularcell specific vector is described by Sato et al. (Proc. Natl. Acad SciUSA, 94:3058-63(1997)

dsRNAi

Another agent for reducing or inhibiting expression of the CD24 gene andproduction of the polypeptide in the T cells of human subjects is adouble stranded oligonucleotide or polynucleotide known as dsRNAi. Onestrand of the dsRNA comprises a CD24 sense sequence; while the otherstrand comprises a CD24 antisense sequence. Preferably, the dsRNAifurther comprises a linker connecting the antisense sequence to thesense sequence. Preferably, the CD24 sense and anti-sense sequences arefrom 19-30 bases in length. The linker is at least 10 bases in length,and preferably, from 10-20 bases in length. The CD24 dsRNAi preventsaccumulation of CD24 mRNA in the transformed cells, most likely througha post-transcription gene silencing method known in the art asdouble-stranded RNA interferences.

dsRNAi can be synthesized using standard techniques. For examplesingle-stranded RNA corresponding to the sense CD24 sequence, and singlestranded RNA corresponding to the antisense CD24 sequence can besynthesized according to methods known in the art. The single strandedRNAs can then be annealed in vitro by methods known in the art, toproduce the dsRNA. To increase the stability of the dsRNAi, severalnucleotide with de-oxyl-nucleotide can be incorporated at the 3′ of theoligonucleotides.

Alternatively, an expression vector comprising a polynucleotide ornucleic acid encoding CD24 dsRNAi is introduced into the subject's Tcells or, the subject's vascular endothelial cells, or both. Suchpolynucleotide comprises a sequence which encodes a sense CD24 RNAcoding sequence and an antisense CD24 RNA coding sequence and a linkersequence which links the sense CD24 RNA coding sequence to the antisenseCD24 RNA coding sequence. The expression vector further comprises apromoter operatively linked to the CD24 dsRNAi coding sequence.

Targeting of the CD24 Antisense and dsRNAi Molecules

In accordance with the present method, targeted regions of the CD24 geneand CD24 mRNA include not only the coding region for the CD24polypeptide, but also the 5′-untranslated region, the 3′-untranslatedregion, the 5′ cap region, intron regions and intron/exon or splicejunction regions of the targeted nucleic acid. The functions ofmessenger RNA to be interfered with include all vital functions such astranslocation of the RNA to the site for protein translation, actualtranslation of protein from the RNA, splicing or maturation of the RNAand possibly even independent catalytic activity which may be engaged inby the RNA. The overall effect of such interference with the CD24 mRNAfunction is to cause interference with expression of the CD24polypeptide.

Although numerous areas can be targeted for anti-sense and dsRNAimolecules, there is a significant advantage to target areas in which thesequence is completely conserved between CD24 in mouse and man. In thisway, the CD24 anti-sense and dsRNAi molecules can be screened for bothefficacy and toxicity in preclinical models before they are used forhuman clinical trials. A comparison between human and mouse CD24 cDNAsequence, as listed in FIG. 14, revealed 8 areas within the 2.1 kb areasof human CD24 that can be as targets for antisense and dsRNAi molecules.

Delivery of the CD24 Antisense and dsRNAi Molecules

Single-stranded CD24 anti-sense oligonucleotides and dsRNAi moleculescan be introduced into the subject's cells, including but not limited toT cells, vascular endothelial cells, or both. The molecules areintroduced into the cells either ex vivo or in vivo. “Ex vivo” meansthat these molecules are introduced into the T cells or endothelialcells outside the body of the subject from whom the T cells orendothelial cells are obtained. The cells are then re-introduced backinto the subject. For in vivo delivery to these target cells, the CD24antisense and dsRNAi molecules are introduced into the subject byinjection. Preferably, the injection is intravenous, or intralesionalinjection (as in the case of rheumatoid arthritis).

Delivery of the CD24 antisense or CD24 dsRNAi encodingpolynucleotide-promoter construct into the subject may be either direct,in which case the subject is directly exposed to the construct orconstruct-carrying vector, or indirect, in which case cells are firsttransformed with the construct in vitro, then transplanted into thepatient. The latter method is referred to as cell-based gene-therapy.

A retroviral vector may be used to deliver the CD24 antisense and CD24dsRNAi encoding construct (see Miller et al., 1993, Meth. Enzymol.217:581-599). Retroviral vectors have been modified to delete retroviralsequences that are not necessary for packaging of the viral genome andare maintained in infected cells by integration into genomic sites uponcell division. More detail about retroviral vectors can be found inBoesen et al. (1994) Biotherapy 6:291-302,: Clowes et al, (1994) J.Clin. Invest. 93:644-651; Kiem et al. (1994) Blood 83:1467-1473; Salmonsand Gunzberg (1993) Human Gene Therapy 4:129-141; and Grossman andWilson (1993) Curr. Opin. in Genetics and Devel. 3:110-114.

A lentiviral vector (Science. 1996 Apr 12;272(5259):263-7.) can also beused to deliver genes that encode the antisense drug either in vivo orto ex vivo cells. Unlike a typical retroviral vector, the lentiviralvector can be used to deliver gene to non-dividing cells.

Alternatively, liposomes may be employed to deliver the CD24 antisenseand dsRNAi encoding constructs to target tissues using methods known inthe art. The liposomes may be constructed to contain a targeting moietyor ligand, such as an antigen, an antibody, or a virus on their surfaceto facilitate delivery to the appropriate tissue. For example, liposomesprepared with ultraviolet (UV) inactivated Hemagglutinating Virus ofJapan (HVJ) may be used to deliver DNA to selected tissues (Morishita,et al.). The liposomes may also be surface-coated with phospholipid-polyethyleneglycol conjugates, to extend blood circulation time andallow for greater targeting via the bloodstream. Liposomes of this typeare well known. A variety of liposome have been described in the art todeliver double-stranded nucleotide or naked DNA into cells, both for exvivo cells, or for in vivo delivery.

Receptor-mediated endocytic pathways for the uptake of DNA may permitthe targeted delivery of the CD24 antisense and dsRNAi encodingconstructs to specific cell types in vivo. Receptor-mediated methods ofpolynucleotide delivery in vivo involve the generation of complexesbetween vectors and specific polypeptide ligands that can be recognizedby receptors on the cell surface.

For general reviews of the methods of in vivo polynucleotide delivery(also referred to as gene therapy), see Goldspiel et al (1993) ClinicalPharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev(1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, (1993)Science 260:926-932; and Morgan and Anderson (1993) Ann. Rev. Biochem.62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly known inthe art of recombinant DNA technology which can be used are described inAusubel et al. (eds.) (1993) Current Protocols in Molecular Biology,John Wiley & Sons, NY; and Kriegler (1990) Gene Transfer and Expression,A Laboratory Manual, Stockton Press, NY.

Transgenic and Knock-In Mouse Models to Test the Effect of CD24 BlockersIn Vivo

Since the major cell type in the CNS that expresses HSA is the brainvascular endothelial cells, transgenic vectors that give specificexpression of human CD24 in both T cells and vascular endothelial cellsare used to prepare the transgenic mice. In one preferred embodiment Tcell-specific expression is achieved using a transgenic vector comprisedof human CD24 open reading frame and the proximal lck promoter andvascular endothelial cell specific expression is achieved using atransgenic vector comprised of human CD24 open-reading frame and the TieII promoter, as described in Proc. Natl. Acad Sci USA, 94:3058-63(1997).To avoid interference by the endogenous HSA, the transgenic vector isinjected into the fertilized embryos from mice with a targeted mutationof mouse CD24 as described in J. Exp. Med. 185: 251-262, 1997.Alternatively, the transgenic mice expressing the CD24 gene can be bredto the CD24 (−/−) mice to avoid expression of endogenous CD24. Thetissue specificity of the transgene expression is verified withanti-CD24 mAb, which is available from Pharmingen (San Diego, Calif.),by flow cytometry and immunohistochemistry according to establishedprocedure.

Alternatively, human CD24 knock-in mice can be developed to screen fortherapeutic agents targeted at the human CD24 antigen. The majoradvantage of the knock-in mice is that all the cells that express murineCD24 can be rendered to express human CD24 genes, and as such, theknock-in mice are more relevant for testing the efficacy and safety ofdrugs targeted at the human CD24 antigens. Since mouse and human CD24protein have identical amino acid sequence in the signal peptide region(encoded) regions, the CD24 knock-in mouse can be made by replacing onlythe coding region in exon 2. Moreover, since a major portion of thecoding region encodes for GPI-cleavage signal peptide that will beremoved from the mature protein, replacement of the mouse HSA/CD24 withhuman CD24 can be achieved simply by replacing the 81 by region thatencode for the core region of mature mouse CD24 protein with a 96 byregion that encodes for the core region of mature human CD24 protein.Replacement of some human CD24 protein amino acids with murinecounterpart may be tolerated if such replacement does not change thebinding activity of these molecules to anti-human CD24 antibodies, andfunctional ligands of human CD24 protein. For convenience of cloning,the replacement can be significantly larger than proposed region. Oncethe construct is produced, it is used to transfect mouse embryonic stem(ES) cells, to select for transfectants in which at least one allele ofthe CD24 gene is replaced the construct through homologousrecombination. The recombinant alleles can be screened by PCR and/orSouthern blot according the established procedures. The recombinant EScells are tested for functionality of the recombinant allele. Once thisis verified, the ES cells are used to produce chimera mice. Furtherbreeding yield mice that are homozygous for the knock-in alleles, whichexpress human CD24 gene on cells that had been programmed to expressCD24.

The transgenic and knock-in mice produced as described above are used toscreen drugs targeted at the human CD24 molecules. One example is toscreen for drugs which inhibit or ameliorate autoimmune conditions suchas multiple sclerosis and diabetes. The most suitable murine model formultiple sclerosis is EAE, which is induced by immunizing mice with MOGaccording to a established procedure.

The preferred method for testing drugs targeted at CD24 for diabetes isto breed the human CD24 transgenic mice with non-obese diabetic (NOD)mice to cross the transgene to NOD background. The drugs targeted at HSAor its homologue are administrated at approximately 2-3 weeks todetermine their 1D50 in the reduction of insulitis and spontaneousdiabetes.

Blocking Binding of Autoreactive T Cells to Endotheial Cells In Vitro orIn Vivo

The present invention also relates to a method of blocking binding ofautoreactive T cells to endothelial cells, in vitro or in vivo. In oneaspect, the method comprises contacting the endothelial cells with asufficient amount of HSA or a fusion protein comprising HSA to inhibitinteraction of the autoreactive T cells with HSA molecules present onthe surface of the endothelial cells. The cells may be in vitro, i.e.,in tissue culture, or in vivo, i.e., in the body of a mammal. Blockinginteraction between the endothelial and T cells in vitro is achieved byadding the protein to a chamber that contains both T cells andendothelial cells. The amount of T cells bound to a monolayer ofendothelial cells in the presence or absence of HSA protein isquantified either by counting the number of cells attached, or by othermethods to quantify the number of T cells that were labeled prior toadding to the monolayer.

Interaction between the endothelial and autoreactive T cells in vivo isinhibited by injecting the protein intravenously. To quantify the extentof inhibition fluorescent labeled T cells are administered to an animaland the rolling of T cells along the blood vessel is measured usingestablished procedures known in the art.

In another aspect, interaction of autoreactive T cells with vascularendothelial cells is blocked or reduced by inhibiting or reducingexpression of the CD24 polypeptide in the autoreactive T cells, theendothelial cells or both. Expression of the CD 24 polypeptide in thetarget cells is accomplished by introducing CD24 antisenseoligonucleotides or CD24 dsRNAi into the target cells, or alternatively,transfecting these cells with a polynucleotide which encodes the CD24antisense oligonucleotide or CD24 dsRNAi. As used herein, transfect,refers to introduction of a polynucleotide into the cell where thepolynucleotide may be incorporated into the genome of the cell,converted into an autonomous replicon, or transiently expressed. Thetransfection can be in vivo or ex vivo. “Ex vivo transfection” meansthat transfection occurs outside the body of the subject from whom thetarget cells were obtained. “In vivo transfection” means transfection ofthe target cells within the body of the subject.

All references cited herein are specifically incorporated herein intheir entirety.

EXAMPLES

The following examples are for illustration only and are not intended tolimit the scope of the invention.

Example 1 Treatment of Animals with Experimental AutoimmuneEncephalomyelitis with HSAIg Methods

Mice Wild type C57BL/6 mice (WT) were purchased from the National CancerInstitute (Bethesda, Md.). Mice homozygous for the disrupted HSA(produced with ES cells from C57BL/6 mice) (18) (24) or CD28 (25)(backcrossed to C57BL/6 for more than 8 generations) locus have beendescribed before and are maintained at the animal facilities of the OhioState University Medical Center. HSA transgenic mice (HSATG) have beendescribed previously (See Zhou, Q., Wu, Y., Nielsen, P. J., and Liu, Y.1997. Homotypic interaction of the heat-stable antigen is notresponsible for its co-stimulatory activity for T cell clonal expansion.Eur J Immunol. 27: 2524-2528, which is specifically incorporated hereinby reference.) and have been backcrossed to C57BL/6j background for morethan 5 generations. Mice with HSA exclusively expressed on the T celllineage (HSATG/HSA(−/−)) were generated by crossing HSATG with theHSA(−/−) mice.

Induction and clinical evaluation of EAE The immunogen, MOG peptide35-55 of rat origin (MEVGWYRSPFSRVVHLYRNGK), was synthesized by ResearchGenetics, Inc. (Huntsville, Ala., USA). The purity of the peptidewas >90%. Mice of 8-12 wks of age were immunized subcutaneously with 200μg MOG peptide in complete Freund's Adjuvant (400 μg of Mycobacteriumtuberculosis per ml) in a total volume of 100 μl. They received 200 μgof Pertusis toxin (List Biological, Campbell, Calif.) in 200 μl PBS inthe tail vein immediately after the immunization, and again 48 hourslater. The mice were observed every other day and scored on a scale of0-5 with gradations of 0.5 for intermediate scores: 0, no clinicalsigns; 1, loss of tail tone; 2, wobbly gait; 3, hind limb paralysis; 4,hind and fore limb paralysis; 5, death. T cell proliferation assayDraining lymph node cells were isolated 10 days after immunization.5×10⁵ cells/well were stimulated with given concentrations of MOGpeptide in the presence 6×10⁵ cells/well of irradiated (2,000 rad)syngeneic splenocytes for 60 hours. The cultures were pulsed with³H-thymidine (1 μCi/well; ICN Pharmaceuticals Inc., Costa Mesa, Calif.USA) for another 12 hours, and incorporation of 3H-thymidine wasmeasured in a liquid scintillation P-plate counter.

ELISpot assay to evaluate frequencies of T cells that produce IFN-γ,IL-2 and IL-4 upon restimulation with MOG peptide in vitro The antibodypairs and the procedures have been described (20), except that the MOGpeptide was used for stimulation at 10 μg/ml. The numbers presented arethose of cytokine producers per million of draining lymph node cells.

Histology

Mice were sacrificed by CO₂ inhalation. Spinal cords were removed byinsufflation and fixed in 10% formalin/PBS. Paraffin sections wereprepared and stained with hematoxylin and eosin. Neurological lesionswere graded on each of the 10 cross sections per spinal cord, accordingthe following criteria: 0, no infiltrate; 1, 3 or less focal meningealinfiltrates; 2, more than 3 focal meningeal infiltrates; 3, up to 5perivascular infiltrate foci in the parenchyma with involvement of lessthan 5% of the white matter; 4, 5-10 perivascular foci in the parenchymaor invasions involving 5-25% the white matter; 5, more than 10perivascular foci or diffuse infiltration involving more than 25% of thewhite matter.

Passive Transfer of EAE

Groups of 8-10 WT and HSA(−/−) mice were immunized with 200 μg of MOGpeptide subcutaneously. At 10 days after immunization, draining lymphnodes were harvested and stimulated at 4×10⁶/m1 in Click's EHAA mediumsupplemented. with 15% fetal calf sera, 5% IL-2 supematant, and 50 μg/mlof MOG peptide for 4 days. 1×10⁸ cells were injected i.p. into eachrecipient mouse that had been γ-irradiated (550 rad) 1 h earlier.

Preparation of Fusion Protein and Treatment of EAE z

The HSA fragment encoding the signal peptide and the mature proteinsequence were amplified by PCR, using GGA AAG CTT ATG GGC AGA GC, SEQ IDNO.:6, as forward primer, CGA GAT CTC TGG TGG TAG CG , SEQ ID NO.:7, asreverse primer, and HSA cDNA as template. The PCR products were digestedwith Hind III and Bgi II enzymes and were ligated to Hind III and XbaI-digested pCDM8 vector (Invitrogen, San Diego) and a Xba I and BarnHI-treated DNA fragment encoding human IgG1 Fc, which were amplified byPCR using CAG GGA TCC CGA GGG TGA GTA CTA AGC TAG CTT CAG CGC TCC TGCCTG, SEQ ID NO.:7, as forward primer and CTT CGA CCA GTC TAG AAG CAT CCTCGT GCG ACC GCG AGA GC, SEQ ID NO.:8, as reverse primer, and DNA fromhuman peripheral blood as template. The construct was verified by DNAsequencing and was used to transfect the Chinese Hamster Ovary cellline. The cells that secreted HSAIg fusion protein were amplified inDMEM containing 5% fetal calf serum until confluence. The cellmonolayers were washed with serum-free medium and cultured in optimal Mmedium for 72 hours. The supernatants were collected and the HSAIg waspurified using a protein G column according to the manufacturer'sprotocol. The purity of the protein was verified by SDS PAGE.

Results

To test if HSA is essential for the development of EAE, we immunizedC57BL/6 wild-type (WT), and HSA- or CD28-deficient mice with myelinoligodendrocyte glycoprotein (MOG) peptide AA35-55 in conjunction withcomplete Freund's adjuvant and pertusis toxin. As shown in FIG. 1 a,wild-type mice developed acute EAE within two weeks of peptideimmunization, while those with targeted mutation of either HSA or CD28were completely resistant to EAE induction. Interestingly, whiletargeted mutation of CD28 ablated induction of MOG-specific T cells, asrevealed by proliferative response of draining lymph node cells, that ofHSA had little effect on peptide-specific T cell proliferation (Fig.lb). Moreover, the frequencies of antigen-specific, IL2-, IL4-, andIFNy-producing cells were not altered in HSA(−/−) mice (FIG. 1 c). Theanti-MOG peptide IgG responses were also detected in HSA-deficient mice(data not shown). The differential effects of HSA and CD28 mutations onT cell priming reveal that these genes mediate two distinct checkpointsin the development of EAE: CD28 controls induction of auto-reactive Tcells, while HSA determines their pathogenicity.

Histological analysis of MOG-peptide immunized WT and HSA-confirms theclinical scores. The histological scores were summarized in FIG. 2 a,while representative histology sections were presented FIG. 2 b-d. Asshown in FIG. 2 b, active immunization with MOG peptide induces multipleneurological lesions in the wild-type mice, characterized by multiplelesions with extensive invasion of parenchyma. In contrast, the spinalcords of HSA-KO mice are either devoid of any lesion (FIG. 2 c), or withone or two low grade lesions involving meninges (FIG. 2 d).

We adoptively transferred activated draining lymph node cells to WT andHSA-deficient recipients. As shown in FIGS. 3 and 4, WT T cells inducedsevere EAE in WT recipients within 8 days of adoptive transfer.Interestingly, none of the HSA-deficient recipients developed EAE. ThusHSA expression on T cells alone appears insufficient for EAEdevelopment. Moreover, T cells from HSA-deficient mice failed to inducedisease regardless of HSA gene status in the recipient, which indicatesthat HSA expression on T cells is necessary for EAE development. Theseresults strongly suggest that HSA must be expressed on both host cellsand auto-reactive T cells in order to induce EAE.

To substantiate these observations, we produced mice that expressed HSAexclusively on T cells. We have previously reported the transgenic micein which expression of HSA was under the control of the lck proximalpromoter (HSATG) (22). For this study, We crossed the HSA transgene toHSA-deficient mice to produce mice that expressed HSA exclusively on Tcells (FIG. 5 a). To test if HSA expression on the T cell lineage issufficient for EAE development, we immunized WT, HSA-TG, HSA(−/−) andHSATG HSA(−/−) mice with MOG. As shown in FIG. 5 b, wild-type and HSATGmice developed EAR with essentially identical kinetics, which indicatesthat transgenic expression of HSA on T cells does not prevent theproduction and effector function of self-reactive T cells. Nevertheless,much like HSA (−/−) mice, the mice with exclusive HSA-expression on theT cell lineage failed to develop EAE. These results demonstrated clearlythat HSA expression on T cell lineage alone is insufficient for EAEdevelopment.

The fact that HSA may be a critical checkpoint after activation ofself-reactive T cells suggests a novel approach in treating autoimmuneneurological diseases. Since an anti-HSA mAb was toxic in the EAE modelto address this issue (Data not shown), we produced a fusion proteinbetween the extracellular domain of HSA and the Fc portion of humanIgG1, to block the HSA-mediated interactions. As shown in FIG. 6 a, thefusion protein has an apparent molecular weight of about 10010 undernon-reducing SDS-PAGE. After reduction, it migrated as a 50 kD band. Wetreated mice starting at 8-10 days after immunization with MOG peptide,when MOG-specific T cells response had already expanded in the locallymph nodes. As shown in FIG. 6 b, HSAIg drastically ameliorated EAE.All HSAIg-treated mice recovered substantially earlier than did thecontrol mice. Since MOG-reactive T cells had been activated prior toHSAIg administration, the clinical signs in the treated group mayreflect the fact that some autoreactive T cells had already migratedinto the central nervous system.

HSAIg, a fusion protein consisting of the extracellular domain of mouseHAS and the Fc portion of immunoglobulin, drastically ameliorates theclinical sign of EAE even when administrated after self-reactive T cellshad been expanded. Thus, identification of HSA as a novel checkpoint,even after activation and expansion of self-reactive T cells, provides anovel approach for immunotherapy of autoimmune neurological diseases,such as multiple sclerosis.

Example 2 Production Human CD24Ig Fusion Protein

Fragments of the human CD24 polypeptides lacking the GPI anchor regionare fused with human Ig constant region to form CD24-Ig fusion protein.In one embodiment the CD24 polypeptide fragment comprises the signalpeptide. In another embodiment the CD24 polypeptide fragment lacks thesignal peptide. The fragment of the human CD24 coding sequence issubcloned into vector pIg (from Novagen) Hind III and BamHI sites.Suitable primers useful in subcloning include, but are not limited to,CD24 forward primer (CD24F.H3): G GCC AAG CTT ATG GGC AGA GCA ATG GTG,SEQ ID NO.:9, with Hind III site 5′ to ATG start codon. CD24-Ig reverseprimer (CD24Rig.Bm): GG CCG GAT CCA CTT ACC TGT CGC CTT GGT GGT GGC ATT,SEQ ID NO.10, with Barn HI site and the SD sequence (A CTT ACC TGT, SEQID NO.:11) next to 3′ end of TTKA (direct sequence: ACC ACC AAG GCG, SEQID NO.:12) in Human CD24. The construct is transfected into CHO cells,and the CD24Ig is secreted into the tissue culture medium. CD24Ig ispurified by affinity chromatography using a Protein G column. The clonecompresses CD24 signal peptide, CD24 core peptide and the IgG/Fcportion, but lacks the GPI anchor signaling region.

Example 3 Production of Anti-Human CD24 mAb that Blocks Autoreactive TCells-Initiated Tissue Destruction

Human CD24 coding sequence is subcloned into vector pCDM8 (fromInvitrogen) Hind III and Xho I sites. CD24 forward primer (CD24F.H3): GGCC AAG CTT ATG GGC AGA GCA ATG GTG with Hind III site 5′ to ATG startcodon. CD24 reverse primer (CD24R. Xho): A TCC CTC GAG TTA AGA GTA GAGATG CAG with Xho I site 3′ to TAA stop codon. The CD24 cDNA istransfected into murine 3T3 cells. The 3T3 cell lines that stablyexpress human CD24 molecules are used to immunize syngeneic mice. After2-3 immunization, spleen cells are fused with myeloma AgX865, afterselection with HAT medium the supernatants are screened for anti-humanCD24 mAbs. The antibodies are tested for their ability to block bothadhesion of human T cells to human endothelial cells in vitro, and theirability to block human CD24-mediated T cell trafficking to targettissues, such as the pancreas and the central nervous system using thetransgenic model detailed below.

Example 4 Testing Putative Inhibitors of Multiple Sclerosis with CD24Transgenic and Knock-In Mice

The immunogen, MOG peptide 35-55 of rat origin (MEVGWYRSPFSRVVHLYRNGK,SEQ ID NO.:13), is available from Research Genetics, Inc. (Huntsville,Ala., USA). Mice of 8-12 wks of age are immunized subcutaneously with200 μg MOG peptide in complete Freund's Adjuvant (400 μg ofMycobacterium tuberculosis per ml) in a total volume of 100 μl. Theyreceive 200 μg of Pertusis toxin (List Biological, Campbell, Calif.) in200 Id PBS in the tail vein immediately after the immunization, andagain 48 hours later. The mice are observed every other day and scoredon a scale of 0-5 with gradations of 0.5 for intermediate scores: 0, noclinical signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limbparalysis; 4, hind and fore limb paralysis; 5, death. The putativeinhibitory molecules are injected at 1 week after immunization. Thosethat substantially reduce the clinical score of EAE are selected forfurther testing.

Example 5 Generation of Human CD24 Gene Knock-in Mice

The basic strategy used to produce CD24 gene knock-in mice is to replacepart of murine CD24 gene exon 2 sequence with that of human CD24sequence. We took advantage of the fact that signal peptide, encoded byexon 1 of mouse and human CD24 gene, are identical between mouse andhuman CD24. We therefore replaced only part of the mouse exon 2 sequencewith that of 240 by of human CD24. The construct with the desiredsequence is shown in FIG. 13.

As shown in FIG. 13, arm 1 of the construct comprised of a 2.7 kbfragment of mouse CD24 gene, cloned from 129RI ES cells (Seq ID. 20).The arm 2 of the construct is a chimera gene consisting of the last 256by sequence of CD24 exon 1, first 240 by human CD24 exon 2 sequence andabout 3 kb of mouse CD25 sequence comprising of both remaining exon 2sequence encoding for 3′ untranslated region and 3′ sequence of the CD24gene Seq I.D. 22). The construct is used to transfect ES cells. Therecombinants are screened by procedures established in the art,including PCR and Southern blot. The ES cells with the illustratedknock-in alleles are transfected with plasmid encoding Cre recombinasethat recognize the lox P sequence. Since ES cells expression CD24 gene,as revealed by cell surface flow cytometry, the functionality of theknock-in alleles can be confirmed by cell surface expression of humanCD24. The ES cells with the capacity to express human CD24 are used toproduce chimera mice by blastocyte injection according to techniqueknown in the art. Mice with germ-line transmission are produced bybreeding the chimera mice.

Example 6 Inhibition of CD24 Expression by dsRNAi Technology

Mouse and human CD24 genes are highly homologous. It is thereforepossible to select regions that are identical between mouse and humanCD24 as target for dsRNAi drug. An alignment between Human CD24(XM_(—)099027) and Mouse CD24 (NM_(—)009846) is shown in FIG. 14. Eightregions with a stretch of identical nucleotide that is 17 by or longerare highlighted and as preferred target sequences. Although identitybetween mouse and human is not an essential feature of the dsRNAimolecule, targeting the dsRNAi to identical regions provides a dsRNAiwhich can be used to inhibit expression of both mouse and human CD24genes. As a result, preclinical small rodent models can be used toscreen for the efficacy of dsRNAi molecule in animal disease models, inaddition to cell culture.

CHO cells transfected with either mouse or human CD24 cDNA aretransfected with dsRNAi, produced by in vitro annealing. Briefly, bothsense and antisense RNA corresponding to nt. 46-64 (+1 as translationstarting site) of mouse and human CD24 gene plus two thymidine weresynthesized by a commercial vendors. The sequence of the two strands areas follows: CD24-46/64 iRNA.F: 5′-CUG GCA CUG CUC CUA CCC ATT-3′ (seqID. 16), and CD24-46/64 iRNA.R: 5′-UGG GUA GGA GCA GUG CCA GTT-3′ (seqID 17). Control oligonucleotides were designed based the invertedsequence, as follows invCD24-46/64 iRNA.F: 5′-ACC CAU CCU CGU CAC GGU CTT-′ (seq ID. 18) invCD24-46/64 iRNA.R: 5′-GAC CGU GAC GAG GAU GGGUTT-3′ (seq ID 19). For annealing of siRNA, 20 uM single strands will beincubated in annealing buffer (100 mM KOAc, 30 mM HEPES at p17.4, 2 mMMgAc) for 1 min at 94 degree followed by lh at 37 degree and resultingdsRNA. The resulting dsRNAi is used to transfect CHO cells. At 48 hoursafter transfection, the cells are analyzed for CD24 expression by flowcytometry.

As shown in FIG. 15, transient transfection lead to expression of CD24on about 7% of the CHO cells. Inverted dsRNA reduced expression of CD24some what, although significant number of CHO cells (2%) still expresshigh level of CD24. Importantly, the expression of CD24 is completelyabrogated when the CHO cells are co-transfected with dsRNAicorresponding to human/mouse CD24 sequence. These results revealed thatthe dsRNAi can be used to inhibit expression of CD24.

1.-28. (canceled)
 29. A method for treating a human patient suspected ofhaving, known to have, or predisposed to having multiple sclerosis, saidmethod comprising administering to said patient a pharmaceuticalcomposition comprising an isolated human HSA/CD24 polypeptide fusion anda physiologically acceptable carrier, wherein the HSA/CD24 polypeptidefusion comprises a HSA/CD24 core region, and wherein the HSA/CD24polypeptide fusion is glycosylated.
 30. The method of claim 29, whereinthe HSA/CD24 fusion comprises the HSA/CD24 polypeptide linked by apeptide bond to a tag, wherein the tag consists of 5 to 300 amino acids.